Magnetic circular dichroism studies of exogenous ligand and substrate binding to the non-heme ferrous active site in phthalate dioxygenase

Elizabeth G. Pavel, Laura J. Martins, Walther R Ellis, Edward I. Solomon

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

Background: Mononuclear non-heme iron centers are found in the active sites of a variety of enzymes that require molecular oxygen for catalysis. The mononuclear non-heme iron is believed to be the active site for catalysis, and is presumed to bind and activate molecular oxygen. The mechanism of this reaction is not understood. Phthalate dioxygenase is one such enzyme. Because it also contains a second iron site, the Rieske site, it is difficult to obtain information on the structure of the active site. We therefore used magnetic circular dichroism (MCD) spectroscopy to probe the mononuclear non-heme Fe2+ site in this biodegradative enzyme. Results: The MCD spectrum of the resting enzyme shows features indicative of one six-coordinate Fe2+ site; substrate binding converts the site to two different five-coordinate species, opening up a coordination position for O2 binding. MCD spectra of the corresponding apoenzyme have been subtracted to account for temperature-independent contributions from the Rieske site. Azide binds both to the resting enzyme to produce a new six-coordinate species, showing that one of the ferrous ligands is exchangeable, and also to the enzyme-substrate complex to form a ternary species. The low azide binding constant for the substrate -enzyme species relative to the resting enzyme indicates steric interaction and close proximity between exogerious ligand and the substrate. Conclusions: We have been able to provide some detailed structural insight into exogenous ligand and substrate binding to the non-heme Fe2+ site, even in the presence of the enzyme's [2Fe2S] Rieske center. Further mechanistic studies are now required to maximize the molecular-level detail available from these spectroscopic studies.

Original languageEnglish (US)
Pages (from-to)173-183
Number of pages11
JournalChemistry and Biology
Volume1
Issue number3
DOIs
StatePublished - 1994
Externally publishedYes

Fingerprint

Circular Dichroism
Catalytic Domain
Ligands
Substrates
Enzymes
Azides
Iron
Molecular oxygen
Catalysis
Oxygen
Circular dichroism spectroscopy
Apoenzymes
phthalate dioxygenase
Spectrum Analysis
Binding Sites
Temperature

Keywords

  • NIR MCD
  • non-heme ferrous
  • phthalate dioxygenase
  • Rieske

ASJC Scopus subject areas

  • Organic Chemistry

Cite this

Magnetic circular dichroism studies of exogenous ligand and substrate binding to the non-heme ferrous active site in phthalate dioxygenase. / Pavel, Elizabeth G.; Martins, Laura J.; Ellis, Walther R; Solomon, Edward I.

In: Chemistry and Biology, Vol. 1, No. 3, 1994, p. 173-183.

Research output: Contribution to journalArticle

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AU - Solomon, Edward I.

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AB - Background: Mononuclear non-heme iron centers are found in the active sites of a variety of enzymes that require molecular oxygen for catalysis. The mononuclear non-heme iron is believed to be the active site for catalysis, and is presumed to bind and activate molecular oxygen. The mechanism of this reaction is not understood. Phthalate dioxygenase is one such enzyme. Because it also contains a second iron site, the Rieske site, it is difficult to obtain information on the structure of the active site. We therefore used magnetic circular dichroism (MCD) spectroscopy to probe the mononuclear non-heme Fe2+ site in this biodegradative enzyme. Results: The MCD spectrum of the resting enzyme shows features indicative of one six-coordinate Fe2+ site; substrate binding converts the site to two different five-coordinate species, opening up a coordination position for O2 binding. MCD spectra of the corresponding apoenzyme have been subtracted to account for temperature-independent contributions from the Rieske site. Azide binds both to the resting enzyme to produce a new six-coordinate species, showing that one of the ferrous ligands is exchangeable, and also to the enzyme-substrate complex to form a ternary species. The low azide binding constant for the substrate -enzyme species relative to the resting enzyme indicates steric interaction and close proximity between exogerious ligand and the substrate. Conclusions: We have been able to provide some detailed structural insight into exogenous ligand and substrate binding to the non-heme Fe2+ site, even in the presence of the enzyme's [2Fe2S] Rieske center. Further mechanistic studies are now required to maximize the molecular-level detail available from these spectroscopic studies.

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